Scientists within the Section on DNA Replication, Repair and Mutagenesis (SDRRM) study the mechanisms by which mutations are introduced into DNA. These studies have traditionally spanned the evolutionary spectrum and include studies in bacteria, archaea and eukaryotes. As part of an international scientific collaboration with Andrew Robinson and Antoine van Oijen (University of Wollongong, Australia), Myron Goodman (University of Southern California) and Michael Cox (University of Wisconsin-Madison), we investigated the sub-cellular localization of the translesion synthesis (TLS) polymerase, pol IV, in Escherichia coli. Pol IV is one of three TLS polymerases found in E.coli. All three polymerases are produced at elevated levels in bacteria as part of the SOS response to DNA damage. They have historically been thought to serve as a last resort DNA damage-tolerance mechanism, re-starting replication forks that have stalled at damage sites on the DNA. TLS polymerases are highly error prone: inducing their activities leads to increased rates of mutation (error rates of up to 1 in every 100 nucleotides incorporated into DNA). TLS is an important source of mutations that fuel bacterial evolution. For several species of bacteria, deleting genes for TLS polymerases dramatically reduces rates of antibiotic resistance development in laboratory measurements, and in some cases even reduces infectivity. Many of the drugs used to treat bacterial infections cause an increase in mutation rates as a result of TLS. It remains unclear, however, whether TLS polymerases contribute to resistance by providing damage tolerance, increasing cell survival and thus the chances that a resistant mutant will be found, or by facilitating adaptive mutation selectively increasing mutation rates to speed the evolution of drug resistance. DNA polymerase (pol) IV is thought to be the most abundant TLS polymerase in E. coli. From Western blots, it has been estimated that levels of pol IV increase from approximately 250 molecules per cell in the absence of DNA damage, to 2500 molecules per cell upon activation of the SOS damage response. Pol IV promotes TLS on a variety of different lesion-containing DNA substrates, although its tendency for misincorporation varies with lesion type. Pol IV bypasses adducts to the N2 position of guanines and a variety of alkylation lesions in a mostly error-free fashion. When overexpressed, pol IV induces -1 frameshift mutations in cells treated with alkylating agents. In addition to these lesion bypass activities, pol IV participates in transcription and double strand break-repair repair and contributes significantly to cell fitness in late stationary phase cultures in the absence of any exogenous DNA damage. Pol IV is also reported to be required for formation of adaptive point mutations in the lac operon and was found to be a major determinant in the development of ciprofloxacin resistance in a laboratory culture model. Visualization of pol IV within live bacterial cells would make it possible to better understand how pol IV activity is regulated in response to DNA damage and test proposed models for its TLS activity at replisomes. To do so, we reported a single-molecule time-lapse approach to investigate pol IV dynamics and kinetics in live E. coli cells under normal growth conditions and following treatment with the antibiotic ciprofloxacin, the DNA-damaging agent MMS, or ultraviolet (UV) light. Twenty minutes after treating cells with the DNA-damaging antibiotic ciprofloxacin, we observed a striking increase in pol IV fluorescence, indicative of SOS-dependent up-regulation. During this same period, we observed an increase in formation of punctate foci, consistent with individual molecules of pol IV binding to DNA. The canonical view in the field is that pol IV primarily acts at replisomes that have stalled on the damaged DNA template. In contrast with this view, we observe that only a small proportion (10%) of pol IV foci colocalize with replisome markers. Initially, the proportion of replisomes that contain pol IV tracked with the increasing concentration of pol IV. In the period 90180 min after ciprofloxacin addition, however, colocalization dropped dramatically, despite pol IV concentrations remaining relatively constant. Our data strongly suggest that pol IV is only licensed to carry out TLS at stalled replication forks during the early stages of SOS, whereas it continues to act on substrates outside of replication forks throughout the SOS response. In an SOS-constitutive mutant that expressed high levels of pol IV, few foci were observed in the absence of damage, indicating that access of pol IV to DNA is dependent on the presence of damage, as opposed to concentration-driven competition for binding sites.